Rostislav V. Shevchenko

A review of tissue-engineered skin bioconstructs available for skin reconstruction

Situations where normal autografts cannot be used to replace damaged skin often lead to a greater risk of mortality, prolonged hospital stay and increased expenditure for the National Health Service. There is a substantial need for tissue-engineered skin bioconstructs and research is active in this field. Significant progress has been made over the years in the development and clinical use of bioengineered components of the various skin layers. Off-the-shelf availability of such constructs, or production of sufficient quantities of biological materials to aid rapid wound closure, are often the only means to help patients with major skin loss. The aim of this review is to describe those materials already commercially available for clinical use as well as to give a short insight to those under development. It seeks to provide skin scientists/tissue engineers with the information required to not only develop in vitro models of skin, but to move closer to achieving the ultimate goal of an off-the-shelf, complete full-thickness skin replacement.

Use of a novel porcine collagen paste as a dermal substitute in full‐thickness wounds

A commercially available porcine collagen sheet material has been found previously to be useful as an implant for reconstructive surgery. However, its use as a dermal substitute has been hindered by slow cell penetration and vascularization. A novel paste formulation of this material was investigated for its potential role as a dermal substitute in full‐thickness wounds. A porcine punch biopsy model was initially used to assess the integration of a wide range of material formulations. Selected formulations were then assessed further in a larger wound‐chamber model. Paste formulations were compared with those of sheet and another commercially available dermal regeneration template. The porcine collagen paste became integrated into full‐thickness wounds without rejection and without excessive inflammation. It was detected in wounds up to day 27 postimplantation. Porcine collagen paste was readily infiltrated by host cells by day 2 and supported migrating keratinocytes on its surface. Staining for endothelial cells indicated neovasculature formation as early as day 4 and functional newly formed microvessels were noted at day 7. This was comparable with neovascularization of an alternative and clinically proven dermal regeneration template and was significantly superior to the sheet material formulation at the same time points. Our findings suggest that porcine collagen paste may be suitable as an alternative to current dermal substitutes in full‐thickness wounds.

Characterisation and performance of hydrogel tissue scaffolds

Porosity over a broad range (typically 0.001–300 μm in diameter) of tissue scaffolds provides appropriate conditions for diffusion and adsorption of small molecules and macromolecules, migration of cells through the scaffold, and adequate cell proliferative capacity. Characterisation of pores over this large range poses a problem especially when analysing soft polymer hydrogels, as no one methodology can adequately cover the entire range. This work describes a combined technique used for evaluation of the porous structure of a collagen hydrogel (dermal substitute Integra®) on the basis of NMR-cryoporometry (sensitive to nanopores) and confocal laser scanning microscopy (CLSM) imaging (sensitive to macropores). Thermodesorption of water, diffusion of proteins through a collagen membrane, migration and growth of normal primary human skin fibroblasts, and the interaction kinetics of 3T3 mouse fibroblast cells (using a quartz crystal microbalance) with collagen were analysed with respect to the porous structure of the material. The contribution to the total porosity of pores with a diameter of less than 100 nm is low, at approximately 3–5%, a figure estimated using the methods described above. However, these pores are the main contributor to the specific surface area (S ≈ 120 m2 g−1) as larger diameter macropores, with diameters of 20–200 μm, have a much lower surface area at S ≈ 0.4 m2 g−1 relative to their large pore volume V = 14.4 cm3 g−1.

THE USE OF CONFOCAL LASER SCANNING MICROSCOPY TO ASSESS THE POTENTIAL SUITABILITY OF 3D SCAFFOLDS FOR TISSUE REGENERATION, BY MONITORING EXTRA‐CELLULAR MATRIX DEPOSITION AND BY QUANTIFYING CELLULAR INFILTRATION AND PROLIFERATION

The great many varieties of dermal regeneration materials currently being developed requires that sound in vitro assessments of their potential to be infiltrated with host cells are needed prior to any in vivo studies. The biocompatibility of scaffolds is conventionally determined by indirect biochemical assays as measures of cell number/viability within these materials. A disadvantage of these methods is the failure to provide information pertaining to the spatial distribution of cells in situ. In the case of dermal regeneration, if cells are only present in appreciable numbers on the outer portion of a scaffold, then critical aspects such as extra‐cellular matrix (ECM) production and vascularization will be limited, and the likelihood of successful tissue regeneration low. Thus, direct enumeration of cells within scaffolds, with respect to spatial distribution and time, is required to adequately assess the potential biocompatibility. In this article we demonstrate a systematic approach to the quantification of infiltration and proliferation of primary human dermal fibroblasts into the dermal regeneration scaffold, Integra® (the most frequently used dermal regeneration template in burns patients), using confocal laser scanning microscopy (CLSM). Scaffold samples were placed on top of confluent cell layers and infiltrated by cells migrating from the culture dish against gravity. Cells were then enumerated (by nuclear staining) with respect to their location within the scaffold and over a time period of up to 28 days. Large increases in cell numbers were observed on the surface of the scaffolds, together with measurable increases in cell numbers within their interiors. We also describe the immunofluorescent staining of fibroblasts and ECM components as well as the subsequent use of CLSM to qualitatively assess the potential for scaffold re‐modeling. Of particular note was the cellular deposition of a “front” of fibronectin matching the maximum extent of cell infiltration at each time point. We suggest that this key ECM component is a useful indicator of the extent of cellular re‐modeling of a biomaterial.

An in vitro evaluation of fibrinogen and gelatin containing cryogels as dermal regeneration scaffolds

Macroporous cryogels containing mixtures of two key components of the dermal extracellular matrix, fibrinogen and collagen-derived gelatin, were evaluated for use as dermal tissue regeneration scaffolds. The infiltration of human dermal fibroblasts into these matrices was quantitatively assessed in vitro using a combination of cell culture and confocal laser scanning microscopy. The extent of cellular infiltration, as measured by the number of cells per distance travelled versus time, was found to be positively correlated with the fibrinogen concentration of the cryogel scaffolds; a known potentiator of cell migration and angiogenesis within regenerating tissue. An analysis of the proteins expressed by infiltrating fibroblasts revealed that the cells that had migrated into the interior portion of the scaffolds expressed predominantly F-actin along their cytoplasmic stress fibres, whereas those present on the periphery of the scaffolds expressed predominantly α-smooth muscle actin, indicative of a nonmotile, myofibroblast phenotype associated with wound contraction. In conclusion, the cryogels produced in this study were found to be biocompatible and, by alteration of the fibrinogen content, could be rendered more amenable to cellular infiltration.

A Novel Skin Substitute Biomaterial to Treat Full-Thickness Wounds in a Burns Emergency Care

A novel porcine collagen-based paste dermal substitute to treat full-thickness wounds has been investigated. A thin split-thickness skin graft or autologous cultured keratinocytes have been combined with dermal replacement biomaterial and applied to full-thickness wounds in a porcine wound chamber preclinical experimental model. The data obtained suggest that: (1) dermal substitute biomaterials may improve wound re-epithelialisation when combined with cultured autologous keratinocytes and (2) porcine collagen paste is able to support split-thickness skin graft survival as well as autologous cultured keratinocyte proliferation. These results demonstrate that the novel porcine collagen paste has a potential as a dermal substitute to treat acute full-thickness wounds and an application for the burns emergency care treatment.